土槿乙酸对高糖环境下人视网膜微血管内皮细胞表达血管内皮生长因子的影响

Pseudolaric acid B down-regulates the expression of vascular endothelial growth factor in human retinal microvascular endothelial cells under high glucose environment

目的观察土槿乙酸对高糖环境下人视网膜微血管内皮细胞(human retinal microvascular endothelial cells,HRMEC)表达血管内皮生长因子(vascular endothelial growth factor,VEGF)的影响。方法体外培养HRMEC,根据不同的实验目的将其分为低糖空白对照(PL)组、土槿乙酸对照(P0)组、高糖对照(PH)组以及不同浓度土槿乙酸+高糖(P1、P2、P3)组。采用MTT法检测土槿乙酸作用后HRMEC的增殖情况,实时定量PCR和Western blot检测VEGF的mRNA和蛋白表达以及核转录因子FOXO3a的磷酸化,免疫荧光检测FOXO3a的核转位,染色质共沉淀检测FOXO3a和VEGF启动子的结合。结果PH组HRMEC增殖率和VEGF表达水平明显高于PL组,并且随着时间的延长,HRMEC增殖率和VEGF表达均逐渐增高(均为P<0.05);而经不同浓度土槿乙酸处理后的P1组、P2组和P3组中的HRMEC增殖率及VEGF表达水平与PH组相比,差异也均有统计学意义(均为P<0.05)。Western blot结果显示,P0组HRMEC中FOXO3a磷酸化水平较低,PL组和PH组均有不同程度增高,而P1、P2、P3组随着土槿乙酸浓度的增加,FOXO3a磷酸化水平随之降低。免疫荧光和染色质共沉淀结果显示,P0组HRMEC中FOXO3a在细胞核内含量较多,和VEGF启动子的结合水平较高;而在PL组和PH组,细胞浆中FOXO3a明显增多,核内FOXO3a和VEGF的结合水平低于P0组;P1组、P2组及P3组中细胞核内FOXO3a以及FOXO3a-VEGF DNA复合体明显增多。结论土槿乙酸能抑制高糖条件下HRMEC的增殖,最终激活核转录因子FOXO3a,从而下调HRMEC表达VEGF。

Objective To observe the effect of pseudolaric acid B on the expression of vascular endothelial growth factor(VEGF)in human retinal microvascular endothelial cells under high glucose environment.Methods Human retinal microvascular endothelial cells(HRMECs)were cultured in vitro and divided into low-glucose blank control group(PL group),pseudolaric acid B group(P0 group),high-glucose control group(PH group),high-glucose plus different concentration of pseudolaric acid B group(P1,P2,P3 group)according to the experimental purposes.MTT assay was used to detect the proliferation of pseudolaric acid B-treated cells.Real-time quantitative PCR and Western blot were used to detect the expression of VEGF and phosphorylation of the transcription factor FOXO3a.Immunofluorescence was used to detect the nuclear translocation of FOXO3a.Chromatin coprecipitation was used to detect the binding of FOXO3a to the promoter of VEGF.Results The cell proliferation rate and the expression level of VEGF in PH group were significantly higher than those in PL group,and the cell proliferation rate and the expression level of VEGF increased gradually with the prolongation of time(both P<0.05),while the cell proliferation rate and the level of VEGF in P1,P2 and P3 groups were significantly higher than those in PH group(all P<0.05).Western blot results showed that the phosphorylation level of FOXO3a was lower in P0 group,but PL and PH groups had higher phosphorylation level of FOXO3a.The phosphorylation level of FOXO3a in P1,P2,P3 groups decreased with the increase of pseudolaric acid B concentration.Immunofluorescence and chromatin co-precipitation results also showed that FOXO3a in the P0 group was more abundant in the nucleus and the binding level of FOXO3a to VEGF was high.In PL and PH groups,the cytoplasmic FOXO3a increased significantly,and the binding level of FOXO3a to VEGF in nucleus was lower than that in the P0 group,but increased significantly in the P1,P2,P3 groups.Conclusion Pseudolaric acid B can inhibit the proliferation of HRCECs grown under high glucose environment,and ultimately activate nuclear transcription factor FOXO3a,thereby down-regulating the expression of VEGF.